Cytidine deaminases (CDAs) represent a novel class of enzymes involved in pyrimidine metabolism in both lower and higher organisms. The fundamental cytidine deaminase reaction requires a cytidine deaminase with bound Zn2+, which serves as a Lewis acid in a reaction that activates a water. The activated water serves as a nucleophile that attacks the C4 (sp2) position of the cytosine ring leading to a transition state that proceeds through a tetrahedral intermediate (sp3). A subsequent change from the enol to keto base tautamer results in an aprotic exocylic oxygen at C4, accompanied by the elimination of ammonia. The product is the uridine nucleoside. The reverse reaction is unfavorable and does not occur to any measurable extent on the enzyme.
The synthesis and inhibitory activity of cytidine deaminase inhibitors have been described in the art (Kim, C. H., et al., J Med Chem, 29(8):1374-80, 1996; Laliberte, J. Cancer Chemother Pharmacol, 30(1): 7-11, 1992; Driscoll, J. S., et al., J Med Chem, 34(11):3280-4, 1991; Xiang, S., et al., Biochemistry, 36(16):4768-74, 1997; Xiang, S., et al., Biochemistry, 35(5):1335-41, 1996; Xiang, S., et al., Biochemistry, 34(14):4516-23, 1995; Frick, L., et al., Biochemistry, 28(24):9423-30, 1989.)
Historically the development of the zebularine nucleoside was based on the need for an inhibitory agent to impair the function of cytidine deaminases that metabolize antileukemic nucleosides such as 5-aza-2′-deoxycytidine (5-aza-dC) and cytidine arabinoside (ara-C) (Foubister, V., Drug Discov Today 8(10):430-1, 2003.) Such undesirable cytidine deaminase activity renders these therapeutics ineffective as anti-leukemia drugs. By comparison to 5-aza-dC and ara-C, zebularine is considerably less cytotoxic and more chemically stable, up to pH 12, in aqueous solution (Barchi, J. J., Jr., J. Organic Chem., 57:536-541, 1992; Kelley, J. A., et al., J Med Chem, 29(11):2351-8, 1986). Interest in zebularine as a therapeutic has emerged due to its efficacy as an anti-DNA methyltransferase drug Cheng, J. C., et al., J Natl Cancer Inst, 95(5):399-409, 2003; Zhou, L., et al., J Mol Biol, 321(4):591-9, 2002). In many types of cancers, tumor suppressor genes become inactivated due to abnormal methylation of their promoter regions. Clinical studies have demonstrated that oral administration of zebularine can reactivate genes that naturally suppress cancer through a mechanism that inhibits methylation. However, in animal studies, the efficacy of zebularine in free nucleoside form was limited due to the very high oral doses needed (1 g per kg body weight in mouse models Foubister, V., Drug Discov Today, 8(10):430-1, 2003). Furthermore, zebularine is cytotoxic due to its non-specific action against the enzymes of pyrimidine metabolism and indiscriminate targeting of DNA methyltransferases. What is needed in the art are inhibitors that selectively target cytidine deaminases with the property that inhibition has little or no cytotoxicity.
In this regard, cytidine deaminases active on cytidine or deoxycytidine in RNA or DNA (respectively) and that belong to a family of enzymes known as APOBEC-1 Related Proteins (ARPs) have been shown to play a role in modifying nucleic acid sequences and thereby giving rise to altered protein expression and/or serving as antiviral agents. ARPs modify select cytidines or deoxycytidines in RNA or DNA through targeting sequence specific signals within 5′ and/or 3′ sequences flanking the modified base. The flanking sequences represent a unique context within which the target cytidine of deoxycytidine is embedded.